Successful reproduction in eutherian mammals requires intimate connections between the embryo and uterus, involving adhesion, attachment and placentation, with or without invasion of the endometrium. The complex dialogue between embryo and uterus involves numerous molecules and pathways. The study of the mechanisms driving early embryo-uterus interactions has historically been challenging, particularly discovering the embryo-expressed molecules. Some components of the interactions have been known for decades, but many have been revealed in recent years through innovative transcriptome studies, studies of the contents and functions of extracellular particles, use of in vitro three-dimensional organoids, genetic studies, approaches for dissecting the regulation of maternal immune system cells, and many other approaches. This review focuses on embryo-expressed molecules that support implantation and embryo attachment, implantation and pregnancy establishment. Both classical and recent discoveries are examined, encompassing results from human, murine, livestock and comparative studies. Opportunities for further mechanistic insights are considered. Additionally, the review considers the potential for agricultural and clinical applications of the more recently identified molecular players and mechanisms for enhancing reproductive success or for providing new approaches to contraception.

Post-ovulatory aging (POA) reduces oocyte quality, yet effective interventions remain limited. Here we demonstrate that early intervention with vitamin K2 (menaquinone-4 isoform, MK-4) alleviates POA by inhibiting iron-disorder-induced oxidative stress. Western blot revealed that the expressions of ferroptosis suppressor protein 1 (FSP1) and vitamin K epoxide reductase-like protein (VKORC1L1) were significantly decreased in mouse POA oocytes; however, MK-4 attenuated these changes. Further, MK-4 inhibited ferritinophagy by downregulating NCOA4 and LC3II, markedly restored ferritin level, reduced intracellular Fe[2+] amount, thus attenuating oxidative stress. These results were characterized by reducing lipid peroxidation marker malondialdehyde (MDA) and acyl-CoA synthetase long chain family member 4 (ACSL4) expression, decreasing γ-H2AX and 8-Hydroxy-2'-deoxyguanosine (8-OHdG), and enhancing expression of the mitochondrial antioxidant enzyme superoxide dismutase 1 (SOD1). Additionally, MK-4 suppressed mitophagy by downregulating PINK1 and Parkin, and upregulating Rab7 improving mitochondrial membrane potential. Consequently, abnormalities in chromosomes, spindles, and the cytoskeleton were significantly ameliorated in POA oocytes receiving early MK-4 intervention. Notably, in vitro-aging oocytes expressed higher FSP1 and VKORC1L1 but lower ACSL4 and MDA versus the in vivo counterparts, suggesting a milder lipid peroxidation in vitro. Collectively, our findings uncover a novel anti-aging mechanism triggered by MK-4, highlighting its potential to establish a preventive strategy for female reproductive aging.

cAMP-dependent protein kinase (PKA) plays a central role in regulating sperm motility in both invertebrates and mammals. The sea mussel Mytilus galloprovincialis provides a valuable model for investigating the molecular mechanisms underlying gamete activation in externally fertilizing organisms. In previous work, several catalytic (C) subunit isoforms of PKA were isolated from this species. Here, we report that one of these isoforms appears to be specifically expressed in spermatozoa, indicating the presence of a sperm-specific C-subunit isoform in a non-mammalian species. The protein was purified from male gonadal tissue and identified by LC-MS/MS as the isoform corresponding to UniProt entry A0A8B6GIP6. Sequence analysis revealed that this mussel C-isoform shares strong homology with mammalian sperm-specific Cα2-isoform within the catalytic core but possesses a unique N-terminal region. Whereas mammalian Cα2 contains a short six-residue N-terminal extension, the mussel sperm isoform features a longer acidic extension of 17 amino acids. Moreover, phosphoproteomic analysis reveals the presence of two phosphorylation sites (Ser5 and Thr30) in the N-terminal tail of the mussel C-subunit that are absent in Cα2. These structural differences suggest specific regulatory mechanisms associated with sperm activation in externally fertilizing bivalves. Immunolocalization studies show that the mussel sperm C-subunit is distributed in the acrosomal region, mitochondria, and along the flagellum, where it is predominantly associated with Rmyt2, a type II regulatory subunit previously characterized in the mussel.

While molecular signature contributes to sperm quality and fertility potential, sperm may lost key molecules during cryopreservation. Since sperm cells are transcriptionally inert and rely on interactions with epididymosomes (epEVs) to acquire molecules, herein, we investigated the effects of the addition of epEVs to the cryopreservation extender on post-thaw sperm quality and fertility potential. Epididymal sperm were obtained from six bulls (n = 6) and a pool of epEVs was formed from the epididymal fluid of 15 bulls. After, a total of 30 × 10[6] sperm was cryopreserved in the presence (epEVs) or not (Control) of PKH67 green-labeled epEVs. Post-thaw, sperm were evaluated for interaction with epEVs by flow cytometry (FC) and fluorescence microscopy (FM); motility, vigor, and morphology by microscopy; membrane integrity by FC and FM; microRNA levels by qPCR; and fertility potential by in vitro embryo production (IVEP). EpEVs-sperm showed interaction with green-epEVs, and higher motility and plasma membrane integrity compared to Control-sperm. Levels of 28 microRNAs were higher or uniquely detected in epEVs-sperm. IVEP with epEVs-sperm resulted in a higher rate of hatched blastocyst. Thus, epEVs-supplemented to the sperm cryopreservation extender improved post-thaw sperm quality and embryo developmental potential. These findings provide new insights into the molecular modulation of sperm fertility.

In-vitro embryo production (IVP) in cattle is limited by the suboptimal competence of oocytes matured outside the follicular environment. While maturation is often achieved, incomplete cytoplasmic maturation may impair embryo development and survival. This study evaluated the influence of follicle size on oocyte cytoplasmic maturation, lipid metabolism, cumulus cell function, and embryo cryosurvival. Oocytes were collected from small (< 6 mm), medium (6-9 mm), and large (10-20 mm) follicles and evaluated at 0, 12, and 24 h of in-vitro maturation (IVM). Organelle distribution, lipid and mitochondrial content and distribution, cortical granule distribution, and transzonal projections (TZPs) were evaluated. Gene expression was assessed in surrounding cumulus cells, and embryo development and cryotolerance were evaluated following fertilization and culture. While organelle patterns did not differ among follicle sizes for mitochondria, lipids, or cortical granules, oocytes from large follicles had lower lipid accumulation by 24 h IVM, and fewer TZPs throughout maturation. Gene expression in cumulus cells from large follicles also had a greater abundance of transcripts involved in steroidogenesis, growth factor signaling, and glucose metabolism midway through maturation. Although cleavage and blastocyst development rates were similar across groups, embryos from large follicle oocytes had higher post-thaw survival and lower lipid content than those from small follicles. These findings demonstrate that follicle size affects key metabolic and structural aspects of oocyte maturation, ultimately influencing embryo quality. Selecting oocytes from larger follicles may improve embryo cryosurvival by promoting more complete cytoplasmic maturation and more efficient lipid regulation during IVM.

Seminal plasma proteins are vital for sperm metabolism, protection against oxidative stress, fertility, and early embryo development. A substantial body of data evidence that seminal plasma proteomes are highly species-specific. However, this information remains dispersed across the literature, hindering comparative analyses and limiting the identification of potential, conserved fertility-related biomarkers. To address this gap, we developed SemProtDB (https://semprotdb.org/), the first centralized database of seminal plasma from domestic mammals. It consolidates data from 28 studies, featuring nearly 18,000 protein entries from 8 major species: Bos taurus, Bubalus bubalis, Capra hircus, Ovis aries, Equus caballus, Equus asinus, Canis lupus familiaris, and Sus scrofa. With more than 1500 proteins manually curated, the database provides a user-friendly interface linked to UniProt, NCBI, STRING, and source publications. SemProtDB represents an unprecedented resource for reproductive biology, facilitating comparative and translational research to better understand the molecular mechanisms underlying fertility across species.

Ovarian transplantation causes a loss of follicular reserve, requiring new strategies to optimize post-transplant follicular growth. This study investigated N-(2-hydroxy-3-piperidin-1-ylpropoxy)pyridine-3-carboximidamide; dihydrochloride (BGP-15), a mitochondrial modulator, as a follicular growth stimulator and a collagen-depositing inhibitor, thereby improving graft quality. In vitro, neonatal mouse ovaries were treated with different concentrations of BGP-15. In vivo, adult mice undergoing ovarian autotransplantation received 10 mg/kg of BGP-15. Follicular quantification, gene expression by qRT-PCR (Sirt1, Sirt3, Nampt, Foxo3a, KitL, Pten, Amh), collagen deposition by Picrosirius assay, neoangiogenesis, macrophage infiltration, biometric parameters, and the estrous cycle were evaluated. In vitro, 0.001 µM BGP-15 induced follicular growth. reducing the percentage of primordial follicles (p < 0.0001) and upregulating Sirt1 (p < 0.0001), Sirt3 (p = 0.026), Nampt (p = 0.002), Pten (p = 0.012), and downregulating Amh (p = 0.031). In vivo, BGP-15 treatment in transplanted mice resulted in an earlier resumption of the estrous cycle (p = 0.0458). After transplantation, BGP-15 improved follicular outcome on day 22, increasing healthy follicles (transitional p = 0.0206; primary p = 0.0125; secondary p = 0.0428) and reducing atretic follicles (p = 0.0381). Treated animals exhibited decreased macrophage numbers (p < 0.0001) and collagen deposition (ovary: p = 0.0465; capsule: p = 0.0344), and increased blood vessel density (p < 0.0001). BGP-15 improves follicular growth and survival in ovarian grafts, coinciding with mitochondrial gene dysregulation and reduced collagen deposition. These findings suggest BGP-15 as a promising candidate for improving follicular outcomes after ovarian transplantation.

Pyroptosis, a proinflammatory form of programmed cell death mediated by the NLRP3 inflammasome, has recently been implicated in Sertoli cell inflammatory injury. Crocin, a water-soluble carotenoid glycoside derived from Crocus sativus L. and Gardenia jasminoides Ellis, exhibits anti-inflammatory, antioxidant, and anti-apoptotic properties, yet its role in Sertoli cell pyroptosis remains unclear. Here, we investigated the pharmacological effects and mechanisms of crocin in regulating TM4 Sertoli cell viability and pyroptosis. Network pharmacology based on oligoasthenospermia (OA)-related candidate targets, together with molecular docking, identified HSP90AA1 as a candidate molecular target associated with crocin-mediated regulation of Sertoli cell pyroptosis. MST analysis further provided target-engagement evidence supporting a direct interaction between crocin and HSP90AA1. In TM4 Sertoli cells, crocin improved cell viability, enhanced Ki-67 expression, and protected against lipopolysaccharide/nigericin-induced pyroptosis, as evidenced by reduced lactate dehydrogenase release, decreased PI-positive cells, and downregulated expression of IL-1β, IL-18, NLRP3, Caspase-1, and GSDMD. Moreover, under HSP90AA1-overexpressing conditions, crocin partially reduced pyroptosis-related injury and inflammatory mediator expression, supporting the involvement of HSP90AA1 in crocin-mediated regulation of Sertoli cell pyroptosis. Collectively, these findings indicate that crocin enhances Sertoli cell proliferation and mitigates inflammasome-driven pyroptosis through an HSP90AA1-associated mechanism. These findings should be interpreted as preliminary in vitro evidence for crocin-mediated regulation of Sertoli cell proliferation and pyroptosis, and further validation in animal models and clinical samples is required.

Early embryonic mortality is a major concern to the beef industry, as nearly 32.3% of multiparous cows will not maintain pregnancy past the first sixteen days of gestation. During this period, the uterine environment provides nutrients to support embryonic growth and development. Therefore, our objective was to (1) modulate the luminal metabolome at the onset of conceptus elongation in pregnant beef cows and (2) identify changes in the endometrial epithelial transcriptome during the peri-elongation period. Cows received either TARG: 100 g of a rumen-inert mono- and polyunsaturated fatty acid source plus 60 g of a rumen-protected choline source (n = 50) or CON: 114 g of a saturated fatty acid source (n = 50) daily from 30 days before (d -30) to 30 days after breeding (d 30). Uterine cytobrush was collected from pregnant cows on day 14 for metabolomics (CON = 17, TARG = 15) and on Day 16 for transcriptomics analysis (CON = 6, TARG = 7). On Day 14, the concentration of 23 metabolites (p < 0.1) differed; the concentration of fatty acids, triacylglycerols, hexosylceramides, cholesterol esters, phospholipids, biogenic amines, carboxylic acids, methyl donors, and amino acids was greater in TARG. On Day 16, 675 genes were differentially expressed (FDR ≤ 0.1). Among these genes, 506 genes were upregulated in TARG, involving important genes in pathways such as interferon signaling, fatty acid transport, synthesis, metabolism, immune response, and cell-to-cell adhesion. In conclusion, the dietary supplementation provided herein modulated the uterine environment during the period of conceptus elongation.

Nearly 200 years ago, scientists began examining reproductive cells in calcareous sponges (Calcarea, Porifera). Since then, 238 studies have been published, encompassing descriptions of gametes and embryos (including ultrastructural studies), life history, asexual reproduction, regeneration, and molecular aspects of development. Here, we present a comprehensive synthesis of two centuries of research on the development of Calcarea and review the different steps of the reproductive processes within this class. We analyzed temporal trends, thematic foci, authors, and geographic distribution of the studied species, and the reproductive characteristics of both subclasses (Calcinea and Calcaronea). Bibliographic productivity increased in gradual waves, from classical histology through ultrastructural and experimental embryology to advanced live imaging and molecular approaches. The past half-century has shown acceleration, yet with a persistent geographic bias toward the Mediterranean and North Atlantic species. Molecular datasets are beginning to link phylogenetic context and gene expression during development, but remain fragmentary and lack standardization within the class. We outline priorities for the coming years and identify critical unanswered questions. While significant challenges remain, confronting them actively advances research on Calcarea reproduction and development and firmly establishes it as a distinct field within sponge reproductive science.

Establishing a successful pregnancy relies on effective communication between the mother and fetus. Small extracellular vesicles (sEVs) are pivotal in facilitating intercellular communication, transferring messenger RNA (mRNA), noncoding RNA (ncRNA), and proteins. Considering the prevalence of in vitro fertilization (IVF) in bovine production, despite its lower birth rate, we aimed to evaluate whether there are differences in transcript content in sEVs between pregnancies from artificial insemination fixed-time (FTAI) and in vitro fertilization-embryo transfer (IVF-ET). Uterine fluid was collected on Days 18 and 32 from AI- and IVF-derived pregnancies in beef cattle. sEVs were isolated, and total RNA was extracted for transcriptome and microtranscriptome analysis. Differences in transcript abundance were greatest when comparing Days 18 and 32 within the IVF-ET, suggesting that temporal changes in transcripts are highly influenced by reproductive technique. Gene set enrichment analysis showed 22 gene sets in IVF-ET18 x FTAI18, with mRNAs involved in embryonic pre-adhesion that may impair embryo fixation. In IVF-ET32 x IVF-ET18, target genes of differentially expressed (DE) microRNAs (miRNAs) were enriched in endocytosis pathway, while target genes of DE lncRNAs were enriched in complement and coagulation cascades pathway. In summary, sEVs derived from FTAI and IVF-ET pregnancies have different coding and noncoding transcript content, suggesting failures in maternal-fetal communication post-IVF.

Oocyte and granulosa cells undergo dramatic molecular alterations prior to ovulation, which are critical for acquisition of oocyte developmental competence. These events are orchestrated by a complex bidirectional communication system mediated by the surrounding follicular cells, with follicular fluid reflecting the metabolic output of this system. The composition of follicular fluid is highly dynamic during folliculogenesis, especially following an ovulation-inducing surge of luteinizing hormone, as granulosa cells and theca cells respond and synthesize new metabolites. In the present study, untargeted metabolomics was performed on follicular fluid collected from synchronized Bos taurus cattle at 0, 6, and 22 h after gonadotropin releasing hormone-induced ovulation. High-performance liquid chromatography-mass spectrometry revealed a temporal shift in metabolite abundance across the periovulatory window. Partial least squares discriminant analysis demonstrated clear separation between 0- and 22-h samples, while 6-h samples displayed an intermediate metabolite profile. A total of 489 features were retained after variance-based filtering (interquartile range threshold of 25%), and a covariate-adjusted linear model identified 327 features as significantly altered across time points (p ≤ 0.05). Lipid-associated metabolites, including polyunsaturated fatty acids, oxylipins, and steroid-derived metabolites were enriched prior to induced ovulation while amino acids and related metabolites were progressively enriched from 0 to 22 h. Functional analysis of features that were progressively enriched across the periovulatory window revealed nine significantly affected pathways (false discovery rate < 0.1) associated with amino acid metabolism and biosynthesis, as well as galactose metabolism. These results enhance our understanding of the periovulatory follicular environment and have potential applications for improving in vitro oocyte maturation protocols.

LIM and SH3 protein 1 (LASP1) is a widely expressed scaffold protein associated with actin-rich structures. In Lasp1-knockout mice, reproductive defects such as longer intervals between litters and reduced pup numbers were observed. Consistently, data from the Human Protein Atlas show strong LASP1 expression in spermatids, suggesting a reproductive role. Mammalian fertilization involves sequential processes of sperm activation, capacitation, and acrosome reaction, driven by bicarbonate-mediated PKA activation, CNP-regulated PKG stimulation, F-actin polymerization, Ca[2+]-signaling, and MAPK signaling pathways - culminating in membrane fusion and CD44 cluster formation for interaction with the egg. In this context, we observed differential isoform expression of PKG in human and mice. Our data provide the first evidence of LASP1 presence in the head, midpiece, and tail of human and mouse spermatozoa. During capacitation, LASP1 undergoes dynamic changes, co-localizes with F-actin and stabilizes the equatorial ring segment. In mice, loss of LASP1 did not affect sperm morphology, motility or chemotaxis, but led to changes in protein phosphorylation, incomplete capacitation, and reduced hyaluronan receptor surface expression, revealing a novel role of LASP1 in sperm fertilization.

In this study, we evaluated the effect of a previous exposure of in vitro produced blastocysts to hypothermia (33°C) on their further cryotolerance. In Experiment 1, embryos were exposed to hypothermia for either 6 or 12 h on day 5 or 6 of development, and the blastocyst rates and kinetics were evaluated. In Experiment 2, the transcriptome of embryos exposed to hypothermia on either day 5 or 6 for 12 h was assessed. In Experiment 3, embryos were exposed to hypothermia for 12 h on day 6, vitrified-warmed, and their re-expansion, apoptotic index, and total cell number were assessed. Results showed that hypothermia had no effect on blastocyst rate but reduced the percentage of hatched blastocysts when applied for 12 h. Moreover, hypothermia was associated with changes in the transcriptional profile, particularly in embryos exposed on day 6, including increased expression of the cold-shock-related transcripts RBM3 and CIRBP. Finally, hypothermia on day 6 did not improve embryo re-expansion after cryopreservation but did reduce apoptosis in the surviving embryos. Our results provide insight into transcriptomic responses associated with mild hypothermia in bovine embryos and support the hypothesis that hormetic-like responses may contribute to improved embryo quality after cryopreservation.